Packing material for chromatography having novel characteristic and method for isolation of substance using the same

ABSTRACT

To provide chromatographic packings whereby biological components, etc. which cannot be separated by either ion-exchange chromatography or reversed phase chromatography employed alone can be efficiently separated without deteriorating their activities. Use is made of a packing which contains a charged copolymer and makes it possible to change the effective charge density on the surface of a stationary phase by a physical stimulus while fixing a mobile phase to an aqueous system.

REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/JP99102698 filed May 24, 1999.

TECHNICAL FIELD

This invention relates to a packing which contains a charged (co)polymerand makes it possible to change the effective charge density orhydrophilic/hydrophobic balance on the surface of a stationary phase inan aqueous system by an external signal (for example, temperature), anda novel separation method by which substances such as metal elements,drugs or biological components are chromatographically separated byusing the packing.

BACKGROUND ART

There a great variety of liquid chromatography techniques depending onthe combination of stationary phase with mobile phase and theinteraction systems employed for the separation. Liquid chromatographyis a highly important technique for separating metal elements, isolatingand purifying drugs and separating peptides, proteins, nucleic acids,etc. in the field of biochemistry. In recent years, moreover, attemptshave been made to apply recombinant proteins, etc. produced bybioengineering procedures, which have made remarkable advances, tomedicines. Under these circumstances, there is a growing requirement forefficient separation methods for separating and purifying theseproducts. Chromatographic techniques commonly employed at presentinvolve ion-exchange chromatography, reversed phase chromatography, etc.

In ion-exchange chromatography, separation is carried out by using, as astationary phase, an electrolyte on the surface of an insoluble carrierand irreversibly adsorbing counter ions contained in the mobile phase.As the carrier, silica, cellulose, dextran, styrene/divinylbenzenecopolymer, etc. are widely employed. Carriers having ion-exchange groups(for example, sulfonate, quaternary ammonium) introduced thereinto arecommercially available as ion exchangers. Solute dissociate intocations, anions and amphoteric ions depending on the hydrogen ionconcentration in the solution. When this solution is passed through anion-exchange column, each ion binds to the oppositely charged exchangegroup on the carrier surface competitively with solvent ions, thuscausing distribution between the solution and the ion exchanger surfaceat a certain ratio. The migration rates through the column varydepending on the bond strength and separation is completed by utilizingthis difference in the migration rate. The distribution can be modifiedby some methods. For example, it can be changed by controlling theconcentration of the competitive ion species in the mobile phase.Alternatively, the extent of ionization of the ion-exchange group on thecarrier surface may be varied by changing the hydrogen ion concentrationin the solution. That is to say, it has been a practice in ion-exchangechromatography to separate solutes from each other by controlling theionic strength or the hydrogen ion concentration in the mobile phase tothereby change the elution order of the solutes.

Reversed phase chromatography involves the use of a combination of ahydrophobic stationary phase and a polar mobile phase. Solutes aredistributed between the mobile phase and the stationary phase dependingon the degree of hydrophobicity. In this case, solutes are eluted alsoby changing the degree of hydrophobicity of the solvent in the mobilephase to thereby change the distribution between the mobile phase andthe stationary phase. Since an organic solvent is employed as thesolvent in the mobile phase, it is feared that the activities of thebiological components to be separated might be caused to deterioratedthereby.

In short, solutes are eluted and separated from each other fundamentallyby varying the solvent in the mobile phase both in ion-exchangechromatography and reversed phase chromatography. Accordingly, there isa risk that the activity of the target sample might be damaged by anacid or organic solvent employed in the elution.

When it is intended to separate substances from each other by two ormore chromatographies, each chromatography should be independentlycarried out, since chromatographic mode varies from carrier to carrier.If it is possible to perform ion-exchange chromatography and reversedphase chromatography by using a single carrier and a single physicalstimulus, separation could be completed at an elevated efficiency withina shorter period of time. Moreover, substances which cannot be separatedfrom each other by the conventional techniques can be separated thereby.

There are a great variety of biological components includingcharged-ones and uncharged ones. In general, a compound capable of beingionized is retained, in an unionized state, in a hydrophobic packingowing to hydrophobic interaction. When ionized, however, the hydrophobicinteraction with the hydrophobic packing is weakened. Ion-dissociatablecompounds differing in the dissociation constant can be easily separatedfrom each other owing to the ion-ion interaction with the use of an ionexchanger.

It is generally known that weakly acidic ion exchange resins and weaklybasic ion exchange resins are suitable respectively for separating basicproteins and acidic proteins. It is thus expected that, by introducingion-exchange substituents, ion-exchange chromatography based on ion-ioninteractions becomes usable in separating various substances, which aresimilar to each other in hydrophobicity or molecular weight and thuscannot be separated exclusively by hydrophobic interactions, andbiological molecules such as proteins and nucleic acid oligomers.

However, there has been known hitherto neither any carrier which isusable both in ion-exchange chromatography and reversed phasechromatography when employed alone under one physical stimulus nor oneusable in efficiently separating various substances, which are similarto each other in hydrophobicity or molecular weight and thus cannot beseparated exclusively by hydrophobic interactions, and biologicalmolecules such as proteins and nucleic acid oligomers.

DISCLOSURE OF INVENTION

To solve the above-mentioned problems, the present inventors haveconducted studies and developments from various viewpoints. As a result,they have successfully prepared a novel packing having ion-exchangefunction by copolymerizing poly(N-isopropylacrylamide)(PIPAAm) withpositively charged dimethylaminopropylacrylamide (DMAPAAm) and foundthat this packing is usable both in reversed phase chromatography andion-exchange chromatography, when temperature is properly controlled.They have furthermore found that use of the charged copolymer makes itpossible to control the LCST of the polymer by regulating pH value. Thepresent invention has been completed based on these findings.

The present invention relates to a method for separating substancescharacterized by chromatographically separating said substances with theuse of a packing which contains a charged (co)polymer and makes itpossible to change the effective charge density on the surface of astationary phase by an external stimulus while fixing a mobile phase toan aqueous system.

The present invention further relates to a method for separatingsubstances characterized by retaining the substances in a stationaryphase made of a chromatographic packing chemically modified with apolyalkylacrylamide copolymer having amino, carboxyl, hydroxyl groups,etc., then changing the hydrophilic/hydrophobic balance on the surfaceof the stationary phase by the temperature gradient method wherein theexternal temperature is changed stepwise, and passing the substancesthrough a single mobile phase to thereby separate the same.

The present invention furthermore relates to a chromatographic packingwhich contains a charged (co)polymer and makes it possible to change theeffective charge density on the surface of a stationary phase by aphysical stimulus.

In the chromatographic packing of the present invention, the chargedstate of ion-exchange groups on the surface of a carrier can bereversibly controlled by changing the surface structure of thestationary phase by an external physical stimulus such as a change intemperature. Namely, the present invention provides a stationary phasewhich makes it possible to perform two chromatographic modes, i.e.,ion-exchange chromatography and reversed phase chromatography, at thesame time with the use of a mobile phase which is a single aqueoussolvent (aqueous mobile phase). Moreover, the present invention providesa carrier capable of arbitrarily controlling the charge of ion-exchangegroups on the surface of the carrier (in the case of ion-exchangechromatography) or the hydrophilic/hydrophobic balance (in the case ofthe reversed phase chromatography). The term “aqueous solvent” as usedherein means water alone or aqueous solutions containing inorganic saltsbut free from any organic solvent.

The present invention provides a carrier for separation and purificationcharacterized in that separation is performed by controlling the chargeof ion-exchange groups on the surf ace of the stationary phase byregulating the physical properties or structure around the ion exchangegroups on the carrier surface by a physical stimulus, while fixing themobile phase to an aqueous system. According to the present invention,when the external temperature is lower than the critical temperature,the ion-exchange groups appear on the surface of the carrier. Then thebiological components to be separated undergo interaction with theion-exchange groups followed by separation by the ion-exchangechromatography mode. When the external temperature is higher than thecritical temperature, on the other hand, the surface charge is weakenedand the carrier becomes more hydrophobic. Then, the biologicalcomponents can be separated by the reversed phase chromatography mode.That is to say, the hydrophilic/hydrophobic balance on the surface ofthe carrier can be reversibly and arbitrarily changed by controlling theexternal temperature.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 provides graphs showing the relationship between temperature andretention time in the separation of aspirin, salicylic acid, methylsalicylate and benzoic acid with the use of two packings described inExample 2.

FIG. 2 provides graphs showing the relationship between temperature andretention time in the separation of aspirin, salicylic acid, methylsalicylate and benzoic acid while changing pH value in Example 3.

FIG. 3 provides graphs showing the relationship between temperature andretention time in the separation of aspirin, salicylic acid, methylsalicylate and benzoic acid while changing ionic strength in Example 4.

FIG. 4 provides graphs showing the relationship between temperature andretention time in the separation of aspirin, salicylic acid, methylsalicylate and benzoic acid while changing the polymerization ratio ofIPAAm to DMAPAAm in Example 5.

BEST MODE FOR CARRYING OUT THE INVENTION

The external physical signal to be used in the method of the presentinvention is exemplified by a change in temperature. To alter thephysical properties or structure around ion-exchange groups on thesurface of the packing by changing temperature, for example, atemperature-responsive polymer may be introduced into the surface of thecarrier. Examples of packings of this type include chromatographicpackings chemically modified on the surface of the carrier withalkylacrylamide polymers or copolymers having amino, carboxyl, hydroxylgroups, etc. in the side chains or at the ends. Chemically modifiedpackings are exemplified by silica carriers modified with theabove-mentioned alkylacrylamide polymers or copolymers. To introduceion-exchange groups, carriers may be chemically modified by copolymersof the above-mentioned alkylacrylamides-with comonomers having amino orcarboxy groups.

Examples of the constitutional units of amino-containing polymersinclude dialkylaminoalkyl(meth)acrylamide, dialkylaminoalkyl(meth)acrylate, aminoalkyl (meth)acrylate, aminostyrene,aminoalkylstyrene, aminoalkyl(meth)acrylamide,alkyloxyalkyltrimethylammonium salts and(meth)acrylamido-alkyltrimethylammonium salts. Examples of theconstitutional units of carboxyl-containing polymers include acrylicacid and methacrylic acid, while examples of the constitutional units ofthe sulfonate-containing polymers include (meth)acrylamido-alkylsulfonicacid.

It is preferable that the polyalkylacrylamide to be used in the presentinvention is selected from among poly(N-isopropylacrylamide),polydiethylacrylamide, poly(N-propylacrylamide) andpolyacryloylpyrrolidine and copolymers of the constitutional units ofthese polymers with alkyl (meth)acrylate, as shown by the followingformulae.

[Chemical formula 1]

R₁ R₂ Abbreviation poly(N-isopropylacrylamide) —H

poly(IPAAm) poly(N,N′-diethylacrylamide) —C₂H₅ —C₂H₅ poly(DEAAm)poly(acryloylpyrrolidine)

poly(APy) poly(N-propylacylamide) —H —C₃H₇ poly(PAAm)

Since poly(N-isopropylacrylamide) has a lower limit of criticaltemperature of 32° C., a carrier chemically modified therewith undergoesa large change in the hydrophilic/hydrophobic surface properties at thiscritical temperature. When the surface of a chromatographic packing isgrafted or coated with this polymer, the power of retaining a samplevaries depending on temperature. Thus, the retention behavior can beregulated by controlling temperature without changing the composition ofthe eluate. A lower limit of critical temperature of 32° C. or above canbe achieved by copolymerizing the N-isopropylacrylamide with comonomerswhich are more hydrophilic than isopropylacrylamide, for example,acrylamide, methacrylic acid, acrylic acid, dimethylacrylamide and vinylpyrrolidone. On the other hand, a lower limit of critical temperaturelower than 32° C. can be achieved by copolymerizing theN-isopropylacrylamide with hydrophobic comonomers, for example, styrene,alkyl methacrylate and alkyl acrylate.

The lower limit of critical temperature of polydiethylacrylamide isabout 30to 32° C. At this temperature, this polymer undergoes a changein the surface hydrophilic/hydrophobic nature. Similar to theabove-mentioned case of poly(N-isopropylacrylamide), the power ofretaining a sample can be thus regulated by controlling temperature. Thenovel chromatographic carrier to be used in the present invention isprepared by chemically modifying or coating the carrier with a polymer.The chemical modification can be carried out by two methods, i.e.,surface grafting and radical polymerization. In the case of coating, onthe other hand, the polymer is insolubilized within the applicationtemperature range and then the insolubilized product is employed incoating.

As described above, surface grafting and radical polymerization can beemployed as the chemical modification means by which atemperature-responsive polymer is introduced into a carrier. In thesurface grafting method, a temperature-responsive polymer of a definitesize is first synthesized and then grafted to the carrier. In theradical polymerization method, in contrast thereto, monomer(s) arepolymerized on the surface of the carrier to give a polymer.

Compared with the surface grafting method, the radical polymerizationmethod makes it possible to introduce the temperature-responsive polymerinto the surface of the carrier at a high density. Thus, thehydrophobicity of the surface of the carrier can be elevated and theretention time can be easily controlled. In this case, moreover,non-specific adsorption on the carrier surface due to the interactionwith silica gel can be easily suppressed.

Substances which can be separated by the method of the present inventioninclude metal element (Cu²⁺, Mn²⁺, etc.), drugs (steroids, antipyreticanalgesic agents, etc.) and biological components (peptides, proteins,nucleic acids, etc.). The method of the present invention isparticularly useful in separating various biological components whichcannot be separated by using either ion-exchange chromatography orreversed phase chromatography alone.

EXAMPLES

To further illustrate the present invention in greater detail, and notby way of limitation, the following Examples will be given.

Example 1 1. Synthesis of Polymer

1-1) Poly(IPAAm-DMAPAAm) (DMAPAAm:N,N-dimethylaminopropyl-acrylamide)

1-1-a) Preparation of IPAAm Copolymer Having Carboxyl End

An IPAAm copolymer having a carboxyl end was synthesized in such amanner as to give a molecular weight of 4,000 as a standard. Themolecular weight of the polymer can be designed by controlling theamount of 3-mercaptopropionic acid (MPA) employed as a chain transferagent. To prepare a copolymer having a molecular weight of 4,000, theamount of MPA was regulated so as to give a molar ratioMPA/(IPAAm+DMAPPAm) of 0.028.

Purified monomer IPAAM: 25.0 g. Cationic monomer (5% by mol of DMAPPAmbased on 1.72 g. IPAAm): Radical polymerization initiator (2,2′-azobis(isobutyro- 0.145 g. nitrile) (AIBN): Chain transfer agent(3-mercaptopropionic acid): 0.691 g. DMF (N,N-dimethylformamide): 50 ml.

The above components were fed into a polymerization tube and fixed witha rubber ring provided with a three-way stopcock. The polymerizationtube was introduced into liquid nitrogen, while closing the cock, andcompletely frozen. Next, the cock was opened and the contents of thetube were degassed by using a vacuum pump. After closing the cock again,the polymerization tube was introduced into methanol and the sample inthe tube was completely dissolved. This procedure was repeated thrice(freezing/thawing degassing method). Then the polymerization tubecontaining the completely degassed sample under reduced pressure wasintroduced into a thermostat under shaking at 70° C., and radicalpolymerization was performed for 2 hours to thereby synthesize acopolymer having a carboxyl group at one end. After the completion ofthe reaction, the reaction mixture was cooled to room temperature byallowing to stand. Then the solvent (DMF) was concentrated by distillingat 40° C. under reduced pressure and the residue was dropped intoice-cooled diethyl ether to thereby give a polymer. The polymer thusobtained was taken up by filtration and dried at ordinary temperatureunder reduced pressure overnight. The dried product was dissolved inacetone and purified again with diethyl ether. The polymer thus obtainedwas taken up again by filtration and dried at ordinary temperature underreduced pressure overnight. The obtained polymer was then dissolved inpurified water to give a 5% (w/v) solution. The resultant solution wastransferred onto a dialysis membrane of a fractional molecular weight of500 and dialyzed for 3 days. Thus a highly pure copolymer having auniform molecular weight could be obtained.

1-1-b) Introduction of IPAAm Copolymer into Carrier

(a) Active Esterification (Succinylation) Method

To succinylate the copolymer synthesized above, the molar ratio of thecopolymer: N,N′-dicyclohexylcarbodiimide (DCC): N-hydroxysuccinimide wasadjusted to 1:2.5:2.

The copolymer was fed into a round-bottomed flask and dissolved in ahalf amount (25 to 30 mL) of ethyl acetate. Next, N-hydroxysuccinimideand DCC were added thereto followed by dissolution in the residualethylacetate. The obtained mixture was immersed in ice-water at 4° C.and stirred with a stirrer for 2 hours. Subsequently, it was introducedinto a thermostat at 25° C. and stirred therein overnight. The solutionwas filtered and thus dicyclohexyl urea formed as a by product wasremoved therefrom. After concentrating under reduced pressure, theresidue was purified with diethyl ether. The product thus obtained wastaken up by filtration and dried under reduced pressure. Thesuccinylated copolymer thus obtained was stored in a freezer.

1-1-c) Introduction into Carrier (Silica Gel)

The succinylated copolymer was reacted in three portions withaminopropyl silica gel with the use of 1,4-dioxane as a solvent. Thereaction was carried out at room temperature (25° C.). First, thesuccinylated polymer (1.0 g) was dissolved in 1,4-dioxane (50 mL) andreacted with aminopropyl silica gel (3 g) in a thermostat under shakingovernight. Subsequently, the liquid reaction mixture was filtered andthe precipitate thus obtained and fresh copolymer (1.0 g) were dissolvedin 1,4-dioxane (50 mL) again and reacted overnight. After repeating thisprocedure once again, the product finally taken up by filtration wassufficiently washed with methanol (500 mL) and distilled water (2 mL),dried under reduced pressure and then stored in a desiccator as apacking.

Example 2

1-2) Preparation of PIPAAm Hydrogel Surface

1-2-a) Formation of Gel Layer on Aminopropyl Silica Gel Surface

To introduce a polymerization initiator into aminopropyl silica gel, thefollowing compounds were used.

Aminopropyl silica gel: 5 g. V-501: 3.5 g (12.5. mmol). EEDQ: 6.18 g(25.0 mmol). DMF: 50 ml.

Use was made of V-501 [4,41-azobis(4-cyanovaleric acid) (molecularweight: 280.28)] as a polymerization initiator and EEDQ[N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, molecular weight:247.30] as a condensing agent each in the amount as specified above.These compounds were reacted with aminopropyl silica gel in DMF. Afterbubbling N₂ gas thereinto in the dark for 30 minutes, the reactionvessel was completely charged with N₂ and reaction was carried out byusing an N₂ balloon at room temperature for 6 hours. After thecompletion of the reaction, the mixture was filtered and washed withDMF. Thus, the polymerization initiator:had been introduced into thesurface of the aminopropyl silica gel.

1-2-b) Formation of Surface Gel Layer

Silica gel having V-501 bonded thereto prepared 4 g. in above 1-2-a):IPAAm: 10 g. BIS: 0.27 g. EtOH: 200 ml. DMAPAAm: such an amount as togive a molar ratio to IPAAm of 8:2 or 9:1.

Silica gel, IPAAm, DMAPAA and BIS [N,N′-methylene-bis (acrylamide),molecular weight: 154.17] were dissolved in ethanol. After bubbling N₂gas thereinto in the dark for 1 hour, the reaction vessel was completelycharged with N₂ and reaction was carried out in an oil bath at 70° C. byusing an N₂ balloon for 5 hours, thus forming a gel layer on the surfaceof PIPAAm. After the completion of the reaction, the mixture wasfiltered and washed with methanol and water. The obtained product wasdried under reduced pressure and stored in a desiccator as a packing. Itwas packed into a stainless column and employed in analysis.

Example 3

Aspirin, salicylic acid, methyl salicylate and benzoic acid wereseparated under the following conditions by using columns packed with apositively charged gal (IPAAm:DMAPAAm=8: 2) and IPAAm hydrogel.

Separation Conditions

Column: (1) packed with poly(IPAAm) hydrogel-modified silica;

(2) packed with poly(IPAAm-co-DMAAAm) (8:2) hydrogel-modified silica.

Buffer: Na₂CO₃/NaHCO₃.

pH=9.0.

Ionic strength=0.1 M.

FIG. 1 shows the results. Aspirin could not be separated from benzoicacid by using the column packed with the IPAAm hydrogel. In contrast,these compounds could be separated from each other by using the columnpacked with the positively charged gel (IPAAm: DMAPPAAm=8:2). At 10° C.,in particular, all of the four compounds including charged and unchargedones could be separated from each other within a short period of time ofabout 20 minutes. The order of separation depended on the hydrophobicitydegrees of these compounds. In the cases of salicylic acid and benzoicacid, the retention times were shortened as temperature was elevated.This is seemingly because, when temperature was elevated, the structureand physical properties of the temperature-responsive polymer werechanged and the charge on the carrier surface was thus lowered so as toreduce the interactions between the surface and the solutes. On thecontrary, methyl salicylate (i.e., an uncharged compound) showed anprolonged retention time as temperature was elevated. This is seeminglybecause the temperature-responsive polymer became hydrophobic due toincrease in temperature.

Example 4 Effects of pH Change

Aspirin, salicylic acid, methyl salicylate and benzoic acid wereseparated by the same method as the one of Example 3 but using thecolumn packed with poly(IPAAm-co-dMAPAAm) (8:2) hydrogel-modified silicaof Example 3 and NaHPO₄/H₃C₆H₅O₇ [citric acid H₂O (monohydrate)] as abuffer at pH 7.0. FIG. 2 shows the results.

FIG. 2 indicates that the retention times of all of the substances wereprolonged at pH 7.0, compared with at pH 9.0. This is seemingly becauseanionic compounds would undergo stronger interactions with thepositively charged carrier surface at pH 7.0. These results suggest thatthe retention times of substances to be separated can be controlled byregulating pH value.

Example 5 Effects of Ionic Strength

Aspirin, salicylic acid, methyl salicylate and benzoic acid wereseparated by using a column packed with the poly(IPAAm-co-DMAPAAm) (8:2)hydrogel-modified silica of Example 3 under the following separationconditions.

Separation Conditions

Buffer: NaHPO₄/H₃C₆H₅O₇.

pH=7.0.

Ionic strength=1.0 M and 0.1 M.

FIG. 3 shows the results. As the ionic strength was elevated (0.1 M→1.0M), the retention times of all of the compounds but the uncharged methylsalicylate were shortened, while the retention time of methyl salicylatewas prolonged. This is seemingly because, when the ionic strength waselevated, the protonation of amino groups on the surface of the carrierwas suppressed and the positive charge was lowered, which weakened theinteractions of the carrier surface with the anionic compounds. In thecase of methyl salicylate, the hydrophobicity was elevated with anincrease in the ionic strength and, in its turn, the hydrophobicinteraction was seemingly strengthened.

Example 6 Effect of Polymerization Ratio of IPAAm to DMAPAAm

Aspirin, salicylic acid, methyl salicylate and benzoic acid wereseparated under the following conditions.

Separation conditions Column: (1) packed with poly(IPAAm-co-DMAPAAm)(9:1) hydrogel-modified silica; (2) packed with poly(IPAAm-co-DMAPAAm)(8:2) hydrogel-modified silica. Buffer: NaHPO₄/H₃C₆H₃O₇. pH = 7.0. Ionicstrength = 0.1 M.

FIG. 4 shows the results. The retention times were prolonged with anincrease in the ratio of the positively charged polymer, which indicatesthat retention time can be controlled by regulating the polymerizationratio.

Industrial Applicability

The present invention has the following advantages.

1) The charge of an ion exchanger exposed on the surface of the carriercan be arbitrarily controlled by regulating temperature. Thus separationcan be performed in a single aqueous mobile phase without changing thesolvent in the mobile phase.

2) Due to differences in hydrophobicity and ionic properties, separationcan be carried out by a single operation. Compared with the conventionalmethods wherein two separating operations are needed, therefore, themethod of the present invention is a highly efficient one and gives anelevated yield.

3) the method of the present invention makes it possible to separatebiological components which cannot be separated by either ion-exchangechromatography or reversed phase chromatography employed alone.

4) since neither any acid nor organic solvent is used in the method ofthe present invention, biological components can be separated withoutdeteriorating their activities.

5) compared with the conventional ion exchangers, the packing of thepresent invention can be quickly regenerated.

What is claimed is:
 1. A method for separating substances characterizedby chromatographically separating said substances with the use of apacking which contains a (co)polymer having a charged group in a sidechain or at an end and makes it possible to change the effective chargedensity on the surface of a stationary phase by a physical stimuluswhile fixing a mobile phase to an aqueous system.
 2. The separationmethod as claimed in claim 1, wherein said physical stimulus is a changein temperature.
 3. The separation method as claimed in claim 2, whereinsaid packing is a chromatographic packing chemically modified on thesurface of a carrier with a temperature-responsive polymer.
 4. Theseparation method as claimed in claim 3, wherein said packing is achromatographic packing chemically modified with atemperature-responsive polymer by using a radical polymerization method.5. The separation method as claimed in claim 3 wherein saidtemperature-responsive polymer, with which the surface of the carrier ischemically modified, is a polyalkylacrylamide polymer or copolymerhaving an amino group, a carboxyl group, or a hydroxyl group in the sidechains or at the ends.
 6. The separation method as claimed in claim 5,wherein said polyalkylacrylamide is one selected from amongpoly(N-isopropylacrylamide), poly(N-propylacrylamide),polydiethylacrylamide and polyacryloylpyrrolidine.
 7. The separationmethod as claimed in claim 1, wherein said substances are those selectedfrom among metal elements, drugs and biological components.
 8. A methodfor separating substances characterized by retaining the substances in astationary phase made of a chromatographic packing chemically modifiedwith a polyalkylacrylamide copolymer having an amino group in a sidechain or at an end, a carboxyl group, or a hydroxyl group, then changingthe hydrophilic/hydrophobic balance on the surface of the stationaryphase by a temperature gradient method wherein an external temperatureis changed stepwise, and passing the substances through a single mobilephase to thereby separate the same.
 9. The separation method as claimedin claim 8, wherein said mobile phase is an aqueous solvent.
 10. Theseparation method as claimed in claim 8, wherein saidpolyalkylacrylamide is one selected from amongpoly(N-isopropylacrylamide), poly(N-propylacrylamide),polydiethylacrylamide and polyacryloylpyrrolidine.
 11. The separationmethod as claimed in claim 8, wherein said substances are those selectedfrom among metal elements, drugs and biological components.
 12. Theseparation method as claimed in claim 8, wherein the polyalkylacrylamidecopolymer has a plurality of amino groups, a plurality of carboxylgroups, or a plurality of hydroxyl groups.
 13. A method for separatingsubstances characterized by chromatographically separating saidsubstances with the use of a packing which contains a charged(co)polymer and makes it possible to change the effective charge densityon the surface of a stationary phase by a change in temperature whilefixing a mobile phase to an aqueous system, wherein said packing is achromatographic packing chemically modified on the surface of a carrierwith a temperature-responsive polymer, with which the surface of thecarrier is chemically modified, is a polyalkylacrylamide polymer orcopolymer having a plurality of amino groups, a plurality of carboxylgroups, or a plurality of hydroxyl groups in the side chains or at theends.
 14. The separation method as claimed in claim 13, wherein saidpacking is a chromatographic packing chemically modified with atemperature-responsive polymer by using a radical polymerization method.15. The separation method as claimed in claim 13, wherein saidpolyalkylacrylamide is one selected from amongpoly(N-isopropylacrylamide), poly(N-propylacrylamide),polydiethylacrylamide and polyacryloylpyrrolidine.
 16. The separationmethod as claimed in claim 13, wherein said substances are thoseselected from among metal elements, drugs and biological components.